Radiation resistance remains a significant clinical challenge in treatment of glioblastoma multiforme (GBM). GBM may initially respond to radiotherapy, however, subsequent local recurrence is universal, suggesting insufficient killing of tumorigenic cells by radiation. Emerging evidence suggests that a subpopulation of GBM cells with stem cell-like characteristics, referred to as GBM stem cells, may represent a critical determinant in driving tumor recurrence after radiotherapy. GBM stem cells are more resistant to radiation than matched non- stem GBM cells. Several hundred GBM stem cells are often sufficient to repopulate GBM xenograft tumors in serial transplantation, while non-stem GBM cells fail to do so at numbers several orders of magnitude higher, suggesting adequate eradication of GBM stem cells is required to delay or prevent tumor recurrence. However, there is a considerable gap in understanding the specific mechanisms that protect GBM stem cells against radiation. There is also a lack of effective radiosensitizing strategies that may significantly improve the response of GSCs to radiotherapy. The long-term goal of the research program of the applicant is to identify innovative and transformative therapeutic strategies for improving radiotherapy for GBM and other human cancers. The objective of studies proposed in this application, which is the next step in pursuit of the long-term goal, is to elucidate and targt a mechanistic link between GSC-specific signaling and radioresistance of GBM. The central hypothesis is that the radioresistant phenotype observed in GSCs is due in large part to a Notch- regulated prosurvival signaling network. This hypothesis is formulated on the basis of the preliminary data produced in the applicant's laboratory. The rationale of the proposed research is that a better understanding of the Notch-regulated signaling network has the potential leading to innovative and effective radiosensitizing approaches through combinatorial suppression of multiple pivotal points of this signaling network. Guided by the preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) to delineate the Notch- regulated prosurvival signaling network in GSCs by a complementary combination of genetic rescue experiments and pharmacological approaches; and 2) to rigorously test a drug combination that synergistically targets this Notch-regulated signaling network and effectively represses the tumorigenicity and radioresistance of GSCs in vitro as well as in vivo. It is anticipated that the proposed research, f adequately developed and successfully completed, will generate novel insights into the current paradigm of the radioresistant phenotype observed in GBM and eventually lead to new radiosensitizing approaches for GBM.